Analysis of Circulating Tumor Cells, Fragments, and Debris

ABSTRACT

The methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry and the CellSpotter® fluorescent microscopy imaging system. Analyzing damaged cells has shown to be important. However, there are two sources of damage: in vivo and in vitro. Damage in vivo occurs by apoptosis, necrosis, or immune response. Damage in vitro occurs during sample acquisition, handling, transport, processing, or analysis. It is therefore desirable to confine, reduce, eliminate, or at least qualify in vitro damage to prevent it from interfering in analysis. Described herein are methods to diagnose, monitor, and screen disease based on circulating rare cells, including malignancy as determined by CTC, clusters, fragments, and debris. Also provided are kits for assaying biological specimens using these methods.

PRIORITY INFORMATION

This application is a divisional application of U.S. Ser. No.10/780,399, filed 17 Feb. 2004 which is the U.S. national stage ofPCT/US02/26861 filed23 Aug. 2002, which claims the benefit of claimpriority under 35 U.S.C. 365(c) to U.S. Provisional Applications No.60/314,151 filed 23 Aug. 2001, and No. 60/369,628 filed 3 Apr. 2002. Allof these applications are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Many clinicians believe that cancer is an organ-confined disease in itsearly stages. However, it appears that this notion is incorrect, andcancer is often a systemic disease by the time it is first detectedusing methods currently available. There is evidence that primarycancers begin shedding neoplastic cells into the circulation at an earlydisease stage prior to the appearance of clinical manifestations. Uponvascularization of a tumor, tumor cells shed into the circulation mayattach and colonize at distant sites to form metastases. Thesecirculating tumor cells (CTC) contain markers not normally found inhealthy individuals' cells, thus forming the basis for diagnosis andtreatment of specific carcinomas. Hence, the presence of tumor cells inthe circulation can be used to screen for cancer in place of, or inconjunction with, other tests, such as mammography, or measurements ofPSA. By employing appropriate mononclonal antibodies directed toassociated markers on or in target cells, or by using other assays forcell protein expression, or by the analysis of cellular mRNA, the organorigin of such cells may readily be determined, e.g., breast, prostate,colon, lung, ovarian or other non-hematopoietic cancers.

Thus, in cases where cancer cells can be detected, while there areessentially no clinical signs of a tumor, it will be possible toidentify their presence as well as the organ of origin. Furthermore,based on clinical data, cancer should be thought of as a blood bornedisease characterized by the presence of potentially very harmfulmetastatic cells, and therefore, treated accordingly. In cases wherethere is absolutely no detectable evidence of CTC, e.g., followingsurgery, it may be possible to determine from further clinical studywhether follow-up treatment, such as radiation, hormone therapy orchemotherapy is required. Predicting the patient's need for suchtreatment, or the efficacy thereof, given the costs of such therapies,is a significant and beneficial piece of clinical information. It isalso clear that the number of tumor cells in the circulation is relatedto the stage of progression of the disease, from its inception to thefinal phases of disease.

Malignant tumors are characterized by their ability to invade adjacenttissue. In general, tumors with a diameter of 1 mm are vascularized andanimal studies show that as much as 4% of the cells present in the tumorcan be shed into the circulation in a 24 hour period (Butler, T P &Gullino P M, 1975 Cancer Research 35:512-516). The shedding capacity ofa tumor is most likely dependent on the aggressiveness of the tumor.Although tumor cells are shed into the circulation on a continuousbasis, it is believed that none or only a small fraction will give riseto distant metastasis (Butler & Gullino, supra). Increase in tumor massmight be expected to be proportional to an increase in the frequency ofthe circulating tumor cells. If this were found to be the case, methodsavailable with a high level of sensitivity would facilitate assessmentof tumor load in patients with distant metastasis as well as those withlocalized disease. Detection of tumor cells in peripheral blood ofpatients with localized disease has the potential not only to detect atumor at an earlier stage but also to provide indications as to thepotential invasiveness of the tumor.

However, whole blood is a complex body fluid containing diversepopulations of cellular and soluble components capable of undergoingnumerous biochemical and enzymatic reactions in vivo and in vitro,particularly on prolonged storage for more than 24 hrs. Some of thesereactions are related to immunoreactive destruction of circulating tumorcells that are recognized as foreign species. The patient's immuneresponse weakens or destroys tumor cells by the normal defensemechanisms including phagocytosis and neutrophil activation.Chemotherapy similarly is intended to reduce both cell function andproliferation by inducing cell death by necrosis. Besides these externaldestructive factors, tumor cells damaged in a hostile environment mayundergo programmed death or apoptosis. Normal and abnormal cells(including CTC) undergoing apoptosis or necrosis, have altered membranepermeabilities that allow escape of DNA, RNA, and other intracellularcomponents leading to formation of damaged cells, fragmented cells,cellular debris, and eventual complete disintegration. Such tumor celldebris may still bear epitopes or determinants characteristic of intactcells and can lead to spurious increases in the number of detectedcirculating cancer cells. Whole blood specimens from healthy individualsalso have been observed to undergo destruction of labile blood cellcomponents, herein categorized as decreased blood quality, on prolongedstorage for periods of greater than 24 hours. For example, erythrocytesmay rupture and release hemoglobin and produce cell ghosts. Leukocytes,particularly granulocytes, are known to be labile and diminish onstorage. Such changes increase the amount of cellular debris that caninterfere with the isolation and detection of rare target cells such asCTC. The combined effects of these destructive processes cansubstantially increase cellular debris, which is readily detectable, forinstance, in flow cytometric and microscopic analyses, such asCellSpotter® or CellTracks™, which are described in commonly-owned U.S.Pat. No. 5,985,153 and No. 6,136,182, both of which are incorporated byreference herein.

Detection of circulating tumor cells by microscopic imaging is similarlyadversely affected by spurious decreases in classifiable tumor cells anda corresponding increase in interfering stainable debris. Hence,maintaining the integrity or the quality of the blood specimen is ofutmost importance, since there may be a delay of as much as 24 hoursbetween blood draw and specimen processing. Such delays are to beexpected, since the techniques and equipment used in processing bloodfor this assay may not be readily available in every laboratory. Thetime necessary for a sample to arrive at a laboratory for sampleprocessing may vary considerably. It is therefore important to establishthe time window within which a sample can be processed. In routinehematology analyses, blood samples can be analyzed within 24 hours.However, as the analysis of rare blood cells is more critical, the timewindow in which a blood sample can be analyzed is shorter. An example isimmunophenotyping of blood cells, which, in general, must be performedwithin 24 hours. In a cancer cell assay, larger volumes of blood have tobe processed, and degradation of the blood sample can become moreproblematic as materials released by disintegrating cells, both from CTCand from hematopoietic cells, can increase the background and thereforedecrease the ability to detect tumor cells.

The origin and nature of observed small debris and large clump-likeaggregates are not fully understood, but are believed to involvecellular components or elements originating from target cells,non-target cells, and possibly plasma components. Since CTC can beconsidered immunologically foreign species, normal cellular immuneresponses of the host will occur in vivo even before blood draw. Alsolarge numbers of CTC can be continuously shed from a tumor site, and asteady-state level is maintained in which destruction of CTC equals theshedding rate which in turn depends on the size of the tumor burden (seeJ G Moreno et al. “Changes in Circulating Carcinoma Cells in Patientswith Metastatic Prostate Cancer Correlates with Disease State.” Urology58. 2001).

Various methods are known in this particular art field for recoveringtumor cells from blood. For example, U.S. Pat. No. 6,190,870 to AmCelland Miltenyi teaches immunomagnetic isolation followed by flowcytometric enumeration. However, before immunomagnetic separation, theblood samples are pre-processed using density gradients. Furthermore,there is no discussion of isolating or counting anything other thanintact cells. There is also no visual analysis of the samples.

In U.S. Pat. No. 6,197,523, Rimm et al. describe enumerating cancercells in 100 μl blood samples. The methods use capillary microscopy toconfirm the identity of cells that are found. The methods are specificfor intact cells, and there is no discussion of isolating or enumeratinganything else, such as fragments or debris.

In U.S. Pat. No. 6,365,362 to Immunivest, methods are described forimmunomagnetically enriching and analyzing samples for tumor cells inblood. The methods are specifically directed towards analyzing intactcells, where the number of cells correlates with the disease state. Theisolated cells are labeled for the presence of nucleic acid and anadditional marker, which allows the exclusion of non-target samplecomponents during analysis.

In WO02/20825, Chen describes using an adhesion matrix for enumeratingtumor cells. Briefly, the matrix is coated with specific adhesionmolecules that will bind to cancer cells with metastatic potential. Thematrix can then be analyzed for the presence and type of captured cells.Also described are methods for using the matrix in screening treatments.While steps are taken to discriminate between intact cells and apoptoticor necrotic cells, the apoptotic or necrotic cells are specificallyexcluded from analysis.

In WO00/47998 from Cell Works, two pathways are described for CTC,terminal and proliferative. Both pathways begin with an “indeterminate”cell that progresses, as determined by morphological differences, downeither the terminal or proliferative pathway. A cell in the terminalpathway eventually is destroyed, and a cell in the proliferative pathwaywill form a new metastatic colony as a metastatic tumor. These twopathways were designed to explain morphological differences seen inpatient samples.

Generally, the more resistant and proliferative cells survive toestablish secondary or metastatic sites. In the peripheral circulation,CTC are further attacked in vivo (and also in vitro) by activatedneutrophils and macrophages resulting progressively in membraneperforation, leakage of electrolytes, smaller molecules, and eventualloss of critical cellular elements including DNA, chromatin, etc, whichare essential for cell viability. At a critical point of the cell'sdemise, cell destruction is further assisted by apoptosis. Apoptosis ischaracterized by a series of stepwise slow intracellular events, whichdiffers from necrosis or rapid cell death triggered or mediated by anextracellular species, e.g. a cytotoxic anti-tumor drug. All or some ofthese destructive processes may lead to formation of debris and/oraggregates including stainable DNA, DNA fragments and “DNA ladder”structures from disintegrating CTC as well as from inadvertentdestruction of normal hematopoietic cells during drug therapy, sincemost cytotoxic drugs are administered at near toxic doses.

As shown in WO00/47998, U.S. Pat. No. 6,190,870, and other publications,CTC can circulate as both live and dead cells, wherein “dead” comprisesthe full range of damaged and fragmented cells as well as CTC-deriveddebris. The tumor burden is probably best represented by the total ofboth intact CTC and of damaged CTC, which bear morphologicalcharacteristics of cells. However, some damaged cells, may have largepores allowing leakage of the liquid and particulate cytosolic contentsresulting in a change in the buoyant densities from about 1.06-1.08 togreater than 1.12, or well above the densities of RBC (live and deadcells can be separated at the interface of gradients of d=1.12 and 1.16according to a Pharmacia protocol). Conventional density gradients, asused in # WO00/47998 would lose such damaged CTC in the discarded RBClayer having a range in density of about 1.08 to 1.11. CTC debris thatis positively stained for cytokeratin may also have densities falling inthe RBC or higher ranges, since most intracellular components (with thepossible exception of lipophilic membrane fragments) have densities inthe range of 1.15 to 1.3. Hence, a substantial portion of damaged CTCand CTC debris may be located in or below the RBC layer, and would notbe seen by the density gradient methods in WO00/47998. Some images ofdamaged or fragmented CTC are shown, but it is quite possible the damageoccurred during cytospin or subsequent processing, and is thusartifactual. While the densities of most intact tumor cells may fall inthe WBC region, it is quite likely that damaged CTC in patient sampleshave higher densities that may place them in the RBC layer; outside thereach of gradient techniques.

US Patent Application #2001/0024802 describes methods for bindingfragments and debris to beads. That published application describednumerous possibilities for the density of fragments and debris ofinterest. Upon centrifugation, the beads will be located in a layerabove RBC, because of the pre-determined specific gravity (density) ofthe beads coupled to fragments and/or debris. However, this system isdependent on correctly binding fragments and debris to these beads. Ifany other sample component binds the beads, they may not appear in thedesired location, and subsequently will not be subject to analysis.

Epithelial cells in their tissue of origin obey established growth anddevelopment “rules”. Those rules include population control. This meansthat under normal circumstances the number and size of the cells remainsconstant and changes only when necessary for normal growth anddevelopment of the organism. Only the basal cells of the epithelium orimmortal cells will divide and they will do so when it is necessary forthe epithelium to perform its function, whatever it is depending in thenature and location of the epithelium. Under some abnormal but benigncircumstances, cells will proliferate and the basal layer will dividemore than usual, causing hyperplasia. Under some other abnormal butbenign circumstances, cells may increase in size beyond what is normalfor the particular tissue, causing cell gigantism, as in folic aciddeficiency.

Epithelial tissue may increase in size or number of cells also due topre-malignant or malignant lesions. In these cases, changes similar tothose described above are accompanied by nuclear abnormalities rangingfrom mild in low-grade intraepithelial lesions to severe inmalignancies. It is believed that changes in these cells may affectportions of the thickness of the epithelium and as they increase inseverity will comprise a thicker portion of such epithelium. These cellsdo not obey restrictions of contact inhibition and continue growingwithout tissue controls. When the entire thickness of the epithelium isaffected by malignant changes, the condition is recognized as acarcinoma in situ (CIS).

The malignant cells eventually are able to pass through the basementmembrane and invade the stroma of the organ as their malignant potentialincreases. After invading the stroma, these cells are believed to havethe potential for reaching the blood vessels. Once they infiltrate theblood vessels, the malignant cells find themselves in a completelydifferent environment from the one they originated from.

The cells may infiltrate the blood vessels as single cells or as clumpsof two or more cells. A single cell of epithelial origin circulatingthrough the circulatory system is destined to have one of two outcomes.It may die or it may survive.

Single Cells:

-   1. The cell may die, either through apoptosis due to internal    changes or messages in the cell itself. These messages may have been    in the cell before intravasation or they may be received while in    the blood, or it may die due to the influence of the immune system    of the host, which may recognize these cells as “alien” to this    environment. The results of cellular death are identifiable in    CellSpotter® as enucleated cells, speckled cells or amorphous cells.    These cells do not have the potential for cell division or for    establishing colonies or metastases.    -   Enucleated cells are the result of nuclear disintegration and        elimination—karyorrhexis and karyolysis. They are positive for        cytokeratin, and negative for nucleic acid.    -   The speckled cells are positive for cytokeratin and DAPI and        show evidence of cellular degeneration and cytoplasmic        disintegration. These cells may represent response to therapy or        to the host's immune system as the cytoskeletal proteins        retract.    -   Another dying tumor cell identifiable using CellSpotter® is the        amorphous cell. These cells are probably damaged during the        preparation process, a sign that these may be weaker, more        delicate cells but may also be the result of apoptosis or immune        attack.-   2. A single epithelial malignant cell may have the potential to    survive the circulation and form colonies in distant organs. These    “survivor cells” appear in CellSpotter® as intact cells with high    nuclear material/cytoplasmic material ratio. These cells are    probably undifferentiated and can potentially divide in blood and    form small clumps that may extravasate in a distant capillary, where    the cell may establish a new colony, or it may remain as a single    cell until it extravasates, dividing once it establishes itself in    the new tissue, starting this way a new colony.

Clusters: The primary tumor may shed clusters that enter the circulationas described by B Brandt et al. (“Isolation of prostate-derived singlecells and cell clusters from human peripheral blood.” Cancer Research 56p 4556-4561, 1996). These clusters may remain as clusters and invade adistant tissue or they may become dissociated in the circulation,probably due to differences in pressure in blood or to the immunesystem's intervention. If these cells are dissociated into single cells,they may follow one of the two paths described for single cells above(see 1 and 2). Cluster formations may have an effect in survival byusing the outside cells as a shield that protects the inner cells fromthe immune system.

Once a new colony is established in a new organ, some malignant cellswill continue replicating to form a new tumor. If they reach newcapillaries, the metastasis story may be repeated and a secondarymetastasis occurs.

-   Monitoring of treatment in patients with known carcinomas: A    decrease in the number of tumor cells and/or increase in the    response index may represent a response to patient therapy.    -   Total tumor cells=Dying cells+Survivor cells (TTC=DC+SC)    -   Response Index=dying cells/total tumor cells (RI=DC/TTC).

The higher the response index, the better the response to therapy. A lowresponse index may indicate that the patient is not responding to thetreatment and or that the pt's immune system is not able to handle thetumor load.

A patient who has 50 total tumor cells that were all survivor cells atpre-treatment visit (a RI=0/50=0) and has 50 TTC on follow-up (aftertreatment) visit may have different outcomes depending in the RI. If allthe TTC are SC (i.e. DC=0), there was no response to therapy. If thereare 50 cells but the response index is 40/50=0.8, then either the immunesystem or the therapy is having a negative effect on tumor load,therefore, is a positive response.

-   Decisions in follow-up on patients with known pre-malignancies: When    a pap smear is diagnosed as having cells with atypia or low-grade    intraepithelial lesions, there is always the possibility that these    patients have a more severe abnormality, which cells were missed as    a sampling error. These patients can be colposcoped and biopsied or    they may be asked to return in three months for a repeat pap smear.    If the atypical cells were concurrent with a small focal area of    malignant cells that did not get sampled, the patient will wait 3    months before she gets any follow-up. This may explain why some    pre-malignancies seem to progress quicker than others (misdiagnoses    due to sampling error, causing an artifact in statistics). These are    usually explained as being a more “aggressive” pre-malignancy.    CellSpotter® can be used to help in the decision tree of these    patients. All patients with an abnormal pap (5-10% of the pap smears    in the USA) can immediately be tested for circulating epithelial    cells. Patients with positive tests should be followed-up    immediately and aggressively. Patients with negative results may    wait the three months for the repeat pap. This would simplify the    decision making process for the physician and health professionals    and help the patient trust her follow-up procedure.-   Screening: CellSpotter® image analysis may be used for screening of    the general population with the condition that special, tissue    specific antibodies would be used on a second test on all abnormal    samples. Identification of CTC in a patient may indicate that there    is a primary malignancy that has started or is starting the process    of metastasis. If these cells are identified as of the tissue of    origin with new markers, then organ specific tests, like CT guided    fine needle aspirations (FNA) can be used to verify the presence or    absence of such malignancies. Patients where a primary cannot be    identified may be followed-up with repeat tests after establishing    an individual base line.

In summary, all or some of the above-cited factors can and were found tocontribute to debris and/or aggregate formation that have been observedto confound the detection of CTC by direct enrichment procedures fromwhole blood as disclosed in this invention. The number of intact CTC,damaged or suspect CTC as well as the degree of damage to the CTC, mayfurther serve as diagnostically important indicators of the tumorburden, the proliferative potential of the tumor cells and/or theeffectiveness of therapy. In contrast, the methods and protocols of theprior art combine unavoidable in vivo damage to CTC with avoidable invitro storage and processing damage, thus yielding erroneous informationon CTC and tumor burdens in cancer patients. Finally, the relativelysimple blood test of the present invention described herein, whichfunctions with a high degree of sensitivity and specificity, the testcan be thought of as a “whole body biopsy.”

BRIEF DESCRIPTION OF THE INVENTION

The methods and reagents described in this invention are used to analyzecirculating tumor cells, fragments, and debris. Analysis is performedwith a number of platforms, including multiparameter flow cytometry andthe CellSpotter® fluorescent microscopy imaging system. It is possibleto mimic the damaged CTC that forms fragments and debris. Furthermore,the number of fragments and debris can be correlated back to the numberof circulating tumor cells (CTC). It is also possible to inhibit furtherdamage of CTC between the blood draw and sample processing through theuse of stabilizing agents.

It has been shown herein that the ability to differentiate between invitro damage, caused by specimen acquisition, transport, storage,processing, or analysis, and in vivo damage, caused by apoptosis,necrosis, or the patient's immune system. Indeed, it is desirable toconfine, reduce, eliminate, or at least qualify in vitro damage toprevent it from interfering in analysis.

Herein are described methods to diagnose, monitor, and screen diseasebased on circulating rare cells, including malignancy as determined byCTC, clusters, fragments, and debris. Also provided are kits forassaying biological specimens using these methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—Models of tumor shedding and metastasis. 1 a. shows possiblestages of cells, clusters, and fragments. 1 b. shows the same model withactual images from samples.

FIG. 2—Flow cytometric analysis of immunomagnetically enriched tumorcells from 7.5 ml blood.

FIG. 3—CellSpotter® analysis of a 7.5 ml blood sample from a metastaticprostate cancer patient that was immunomagnetically enriched for tumorcells. The lines of thumbnails correspond to the different dyes used inthe staining process showing tumor candidates stained with cytokeratinPE (green) and DAPI (magenta).

FIG. 4—CellSpotter® classifications of tumor cells isolated from asingle whole blood sample of a patient with metastatic prostate cancerstained with cytokeratin PE (green) and DAPI (magenta).

-   -   A—intact cells    -   B—damaged tumor cells    -   C—tumor cell fragments

FIG. 5—A comparison of the number of obvious CTC to suspect CTC in 20clinical samples.

FIG. 6—CellSpotter® classifications of paclitaxel treated LnCaP cellsspiked into whole blood and isolated then stained with cytokeratin PE(green) and DAPI (magenta).

-   -   A—intact cells    -   B—dying tumor cells    -   C—tumor cell fragments

DETAILED DESCRIPTION OF THE INVENTION

Herein, various terms that are well understood by those of ordinaryskill in the art are used. The intended meaning of these terms does notdepart from the accepted meaning.

The evidence that minimal residual disease in patients with carcinomahas clinical significance is mounting. To effectively monitor minimalresidual disease, a qualitative and quantitative assessment is needed.As the frequency of carcinoma cells in blood or bone marrow is low thelaborious manual sample preparation methods involved in the preparationof samples for analysis often leads to erroneous results. To overcomethese limitations a semi-automated, sample preparation system wasdeveloped that minimize variability and provide more consistent results,as described in commonly-owned pending U.S. application Ser. No.10/081,996, filed 20 Feb. 2002, which is incorporated by referenceherein.

Various methods are available for analyzing or separating theabove-mentioned target substances based upon complex formation betweenthe substance of interest and another substance to which the targetsubstance specifically binds. Separation of complexes from unboundmaterial may be accomplished gravitationally, e.g. by settling, or, bycentrifugation of finely divided particles or beads coupled to thetarget substance. Such particles or beads may be made magnetic tofacilitate the bound/free separation step. Magnetic particles are wellknown in the art, as is their use in immune and other bio-specificaffinity reactions. Generally, any material that facilitates magnetic orgravitational separation may be employed for this purpose. However, ithas become clear that magnetic separation means are the method ofchoice.

Magnetic particles can be classified on the basis of size; large (1.5 toabout 50 microns), small (0.7-1.5 microns), or colloidal (<200 nm),which are also referred to as nanoparticles. The third, which are alsoknown as ferrofluids or ferrofluid-like materials and have many of theproperties of classical ferrofluids, are sometimes referred to herein ascolloidal, superparamagnetic particles.

Small magnetic particles of the type described above are quite useful inanalyses involving bio-specific affinity reactions, as they areconveniently coated with biofunctional polymers (e.g., proteins),provide very high surface areas and give reasonable reaction kinetics.Magnetic particles ranging from 0.7-1.5 microns have been described inthe patent literature, including, by way of example, U.S. Pat. Nos.3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and4,659,678. Certain of these particles are disclosed to be useful solidsupports for immunological reagents.

The efficiency with which magnetic separations can be done and therecovery and purity of magnetically labeled cells will depend on manyfactors. These include:

-   number of cells being separated,-   receptor or epitope density of such cells,-   magnetic load per cell,-   non-specific binding (NSB) of the magnetic material,-   carry-over of entrapped non-target cells,-   technique employed,-   nature of the vessel,-   nature of the vessel surface,-   viscosity of the medium, and-   magnetic separation device employed.    If the level of non-specific binding of a system is substantially    constant, as is usually the case, then as the target population    decreases so will the purity.

As an example, a system with 0.8% NSB that recovers 80% of a populationwhich is at 0.25% in the original mixture will have a purity of 25%.Whereas, if the initial population was at 0.01% (one target cell in 10⁶bystander cells), and the NSB were 0.001%, then the purity would be 8%.Hence, a high the purity of the target material in the specimen mixtureresults in a more specific and effective collection of the targetmaterial. Extremely low non-specific binding is required or advantageousto facilitate detection and analysis of rare cells, such as epithelialderived tumor cells present in the circulation.

Less obvious is the fact that the smaller the population of a targetedcell, the more difficult it will be to magnetically label and torecover. Furthermore, labeling and recovery will markedly depend on thenature of magnetic particle employed. For example, when cells areincubated with large magnetic particles, such as Dynal beads, cells arelabeled through collisions created by mixing of the system, as the beadsare too large to diffuse effectively. Thus, if a cell were present in apopulation at a frequency of 1 cell per ml of blood or even less, as maybe the case for tumor cells in very early cancers, then the probabilityof labeling target cells will be related to the number of magneticparticles added to the system and the length of time of mixing. Sincemixing of cells with such particles for substantial periods of timewould be deleterious, it becomes necessary to increase particleconcentration as much a possible. There is, however, a limit to thequantity of magnetic particle that can be added, as one can substitute arare cell mixed in with other blood cells for a rare cell mixed in withlarge quantities of magnetic particles upon separation. The lattercondition does not markedly improve the ability to enumerate the cellsof interest or to examine them.

The preferred magnetic particles for use in carrying out this inventionare particles that behave as colloids. Such particles are characterizedby their sub-micron particle size, which is generally less than about200 nm (0.20 microns), and their stability to gravitational separationfrom solution for extended periods of time. In addition to the manyother advantages, this size range makes them essentially invisible toanalytical techniques commonly applied to cell analysis. Particleswithin the range of 90-150 nm and having between 70-90% magnetic massare contemplated for use in the present invention. Suitable magneticparticles are composed of a crystalline core of superparamagneticmaterial surrounded by molecules which are bonded, e.g., physicallyabsorbed or covalently attached, to the magnetic core and which conferstabilizing colloidal properties. The coating material should preferablybe applied in an amount effective to prevent non-specific interactionsbetween biological macromolecules found in the sample and the magneticcores. Such biological macromolecules may include carbohydrates such assialic acid residues on the surface of non-target cells, lectins,glyproteins, and other membrane components. In addition, the materialshould contain as much magnetic mass per nanoparticle as possible. Thesize of the magnetic crystals comprising the core is sufficiently smallthat they do not contain a complete magnetic domain. The size of thenanoparticles is sufficiently small such that their Brownian energyexceeds their magnetic moment. As a consequence, North Pole, South Polealignment and subsequent mutual attraction/repulsion of these colloidalmagnetic particles does not appear to occur even in moderately strongmagnetic fields, contributing to their solution stability. Finally, themagnetic particles should be separable in high magnetic gradientexternal field separators. That characteristic facilitates samplehandling and provides economic advantages over the more complicatedinternal gradient columns loaded with ferromagnetic beads or steel wool.Magnetic particles having the above-described properties can be preparedby modification of base materials described in U.S. Pat. No. 4,795,698,No. 5,597,531, and No. 5,698,27, each incorporated by reference herein.

An improved method for making particles is described in U.S. Pat. No.5,698,271. These materials are an improvement over those disclosed inthe '531 patent in that the process includes a high temperature coatingstep which markedly increases the level of coating. Nanoparticles madewith bovine serum albumin (BSA) coating using this process, for example,have a 3-5-fold lower non-specific binding characteristic for cells whencompared to the DC-BSA materials of '531. This decrease in non-specificbinding has been shown to be directly due to the increased level of BSAcoating material. When such nanoparticles were treated so as to removeBSA coating, non-specific binding returns to high levels. It was thusdetermined that a direct relationship exists between the amount of BSAcoated on iron oxide crystal surfaces and the nonspecific binding ofcells. Typically, the non-specific binding of cells from whole bloodwith these particles was 0.3%, which is significantly better than those,produced from '531. Thus, from 10 ml of whole blood there would be about200,000 non-target cells that would also be isolated with the cellstargeted for enrichment.

Since small nanoparticles (30-70 nm) will diffuse more readily they willpreferentially label cells compared with their larger counterparts. Whenvery high gradients are used, such as in internal gradient columns, theperformance of these materials, regardless of size, makes littledifference. On the other hand, when using external gradients, orgradients of lesser magnitude than can be generated on microbead orsteel wool columns, the occupancy of small nanoparticles on cells has asignificant effect. This was conclusively shown to be the case byfractionating DC nanoparticles and studying the effects on recovery.Based on these studies and other optimization experiments, means forfractionating nanoparticles magnetically or on columns was establishedwhere base coated magnetic particles could be prepared that were devoidof excessively small or large nanoparticles. For example, base coatedparticles of mean diameter 100 nm can be produced which contain at besttrace amounts of material smaller than 80 nm or over 130 nm. Similarlymaterial of about 120 nm can be made with no appreciable materialsmaller than 90-95 nm and over 160 nm. Such materials performedoptimally with regard to recovery and could be made sub-optimal by theinclusion of 60-70 nm nanoparticles. The preferred particle size rangefor use in practicing this invention is 90-150 nm for base coatedmagnetic particles, e.g., BSA-coated magnetite.

Based on the foregoing, high gradient magnetic separation with anexternal field device employing highly magnetic, low non-specificbinding, colloidal magnetic particles is the method of choice forseparating a cell subset of interest from a mixed population ofeukaryotic cells, particularly if the subset of interest comprises but asmall fraction of the entire population. Such materials, because oftheir diffusive properties, readily find and magnetically label rareevents, such as tumor cells in blood. For magnetic separations for tumorcell analysis to be successful, the magnetic particles must be specificfor epitopes that are not present on hematopoeitic cells.

A large variety of analytical methods and criteria are used to identifytumor cells, and the first attempts are being undertaken to standardizecriteria that define what constitutes a tumor cell byimmunocytochemistry. In this study, blood samples from prostate cancerpatients were immunomagnetically enriched for cells that expressedEpCAM. Tumor cells were identified by the expression of the cytoskeletalproteins cytokeratin (CK+), the absence of the common leukocyte antigenCD45 (CD45−) and the presence of nucleic acids (NA+) by multicolorfluorescence analysis. Rare events or rare cells can be immunophenotypedby both flowcytometry and fluorescence microscopy. Flowcytometricanalysis excels in its ability to reproducibly quantify even low levelsof fluorescence whereas microscopy has the better specificity asmorphological features can aid in the classification of theimmunophenotypically identified objects. Although there was acorrelation between the number of CTC detected in blood of prostatecancer patients by flowcytometry and microscopy, microscopic examinationof the CK+, CD45−, NA+ objects showed that only few of the objectsappeared as intact cells. This observation agrees with other reportsthat showed apoptosis in a substantial portion of circulating tumorcells.

The terms “biological specimen” or “biological sample” may be usedinterchangeably, and refer to a small potion of fluid or tissue takenfrom a human subject that is suspected to contain cells of interest, andis to be analyzed. A biological specimen refers to the fluidic portion,the cellular portion, and the portion containing soluble material.Biological specimens or biological samples include, without limit bodilyfluids, such as peripheral blood, tissue homogenates, nipple aspirates,colonic lavage, sputum, bronchial lavage, and any other source of cellsthat is obtainable from a human subject. An exemplary tissue homogenatemay be obtained from the sentinel node in a breast cancer patient.

The term “rare cells” is defined herein as cells that are not normallypresent in biological specimens, but may be present as an indicator ofan abnormal condition, such as infectious disease, chronic disease,injury, or pregnancy. Rare cells also refer to cells that may benormally present in biological specimens, but are present with afrequency several orders of magnitude less than cells typically presentin a normal biological specimen.

The term “determinant”, when used in reference to any of the foregoingtarget bioentities, refers broadly to chemical mosaics present onmacromolecular antigens that often induce an immune response.Determinants may also be used interchangeably with “epitopes”. A“biospecific ligand” or a “biospecific reagent,” used interchangeablyherein, may specifically bind determinants. A determinant refers to thatportion of the target bioentity involved in, and responsible for,selective binding to a specific binding substance (such as a ligand orreagent), the presence of which is required for selective binding tooccur. In fundamental terms, determinants are molecular contact regionson target bioentities that are recognized by agents, ligands and/orreagents having binding affinity therefore, in specific binding pairreactions.

The term “specific binding pair” as used herein includesantigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist,lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fcreceptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin andvirus-receptor interactions.

The term “detectably label” is used herein to refer to any substancewhose detection or measurement, either directly or indirectly, byphysical or chemical means, is indicative of the presence of the targetbioentity in the test sample. Representative examples of usefuldetectable labels, include, but are not limited to the following:molecules or ions detectable based on light absorbance, fluorescence,reflectance, light scatter, phosphorescence, or luminescence properties;molecules or ions detectable by their radioactive properties; moleculesor ions detectable by their nuclear magnetic resonance or paramagneticproperties. Included among the group of molecules indirectly detectablebased on light absorbance or fluorescence, for example, are variousenzymes which cause appropriate substrates to convert (e.g. fromnon-light absorbing to light absorbing molecules, or formnon-fluorescent to fluorescent molecules). Analysis can be performedusing any of a number of commonly used platforms, includingmultiparameter flow cytometry immunofluorescent microscopy, laserscanning cytometry, bright field base image analysis, capillaryvolumetry, spectral imaging analysis, manual cell analysis, CellSpotter®analysis, CellTracks™ analysis, and automated cell analysis.

The phrase “to the substantial exclusion of” refers to the specificityof the binding reaction between the biospecific ligand or biospecificreagent and its corresponding target determinant. Biospecific ligandsand reagents have specific binding activity for their target determinantyet may also exhibit a low level of non-specific binding to other samplecomponents.

The phrase “early stage cancer” is used interchangeably herein with“Stage I” or “Stage II” cancer and refers to those cancers that havebeen clinically determined to be organ-confined. Also included aretumors too small to be detected by conventional methods such asmammography for breast cancer patients, or X-rays for lung cancerpatients. While mammography can detect tumors having approximately 2×10⁸cells, the methods of the present invention should enable detection ofcirculating cancer cells from tumors approximating this size or smaller.

The term “enrichment” as used herein refers to the process ofsubstantially increasing the ratio of target bioentities (e.g., tumorcells) to non-target materials in the processed analytical samplecompared to the ration in the original biological sample. In cases whereperipheral blood is used as the starting materials, red cells are notcounted when assessing the extent of enrichment. Using the method of thepresent invention, circulating epithelial cells may be enriched relativeto leucocytes to the extent of at least 2,500 fold, more preferably5,000 fold and most preferably 10,000 fold.

The terms “anti-coagulant” or “anti-coagulating agent” may be usedinterchangeably, and refer to compositions that are added to biologicalspecimens for the purpose of inhibiting any undesired natural orartificial coagulation. An example of coagulation is blood clotting andcommon anti-coagulants are chelating agents, exemplified byethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaaceticacid (DTPA), 1,2-diaminocyclohexane tetraacetic acid (DCTA),ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA), or bycomplexing agents, such as heparin, and heparin species, such as heparinsulfate and low-molecular weight heparins. This may be furthercollectively defined as “clumping’ or “clump formation”. However, suchclumps must be differentiated from “clusters” or aggregates of CTC thatare counted as a single, intact CTC if they meet the classificationcriteria for intact CTC.

Clusters of CTC are believed to have greater proliferative potentialthan single CTC and their presence is thus diagnostically highlysignificant. One possible cause for an increased propensity to establishsecondary metastatic tumor sites may be the virtue of theiradhesiveness. An even more likely cause is the actual size of a CTCcluster; larger clusters will become lodged in small diametercapillaries or pores in bone. Once there, the viability of the cells inthe cluster would determine the chance of survivability at the newmetastatic site.

The ideal “stabilizer” or “preservative” (herein used interchangeably)is defined as a composition capable of preserving target cells ofinterest present in a biological specimen, while minimizing theformation of interfering aggregates and cellular debris in thebiological specimen, which in any way can impede the isolation,detection, and enumeration of targets cells, and their differentiationfrom non-target cells. In other words, when combined with ananti-coagulating agent, a stabilizing agent should not counteract theanti-coagulating agent's performance. Conversely, the anti-coagulatingagent should not interfere with the performance of the stabilizingagent. Additionally, the disclosed stabilizers also serve a thirdfunction of fixing, and thereby stabilizing, permeabilized cells,wherein the expressions “permeabilized” or “permeabilization” and“fixing”, “fixed” or “fixation” are used as conventionally defined incell biology. The description of stabilizing agents herein implies usingthese agents at appropriate concentrations or amounts, which would bereadily apparent to one skilled in cell biology, where the concentrationor amount is effective to stabilize the target cells without causingdamage. One using the compositions, methods, and apparatus of thisinvention for the purpose of preserving rare cells would obviously notuse them in ways to damage or destroy these same rare cells, and wouldtherefore inherently select appropriate concentrations or amounts. Forexample, the formaldehyde donor imidazolidinyl urea has been found to beeffective at a preferred concentration of 0.1-10%, more preferably at0.5-5% and most preferably at about 1-3% of the volume of said specimen.An additional agent, such as polyethylene glycol has also been found tobe effective, when added at a preferred concentration of about 0.1% toabout 5%, more preferably about 0.1% to about 1%, and most preferablyabout 0.1% to about 0.5% of the specimen volume.

A stabilizing agent must be capable of preserving a sample for at leasta few hours. However, it has been shown that samples can be stabilizedfor at least up to 72 hours. Such long-term stability is important incases where the sample is obtained in a location that is distant to thelocation where processing and analysis will occur. Furthermore, thesample must be stabilized against mechanical damage during transport.

Stabilizing agents are necessary to discriminate between in vivo tumorcell disintegration and disintegration due to in vitro sampledegradation. Therefore, stabilizing agent compositions, as well asmethods and apparatus for their use, are described in a co-pendingapplication entitled “Stabilization of cells and biological specimensfor analysis.” That commonly owned application is incorporated byreference herein.

The terms “obvious cells” or “intact cells” may be used interchangeably,and refer to cells found during imaging analysis that contain nucleicacid and cytokeratin. These cells are usually visually round or oval,but may sometimes be polygonal or elongated. The nucleic acid area (i.e.labeled by nucleic acid dye) is smaller than the cytoplasmic area (i.e.labeled by anti-cytokeratin), and is surrounded by the cytoplasmic area.

The terms “suspicious cells”, “suspect cells”, or “fragments” may beused interchangeably, and refer to cells found during imaging analysisthat resemble intact cells, but are not as visually distinct as intactcells. Based on imaging analysis, there are a number of possible typesof suspect cells, including:

-   1. Enucleated cells, which are shaped like obvious cells, are    positively stained for cytokeratin, but negative for nucleic acid;-   2. Speckled or punctate cells, which are positively stained for    nucleic acid, but have irregularly-stained cytokeratin; and-   3. Amorphic cells, which stain positively for cytokeratin and    nucleic acid, but are irregular in shape, or unusually large.    These suspicious cells are of interest in this invention because    they may give additional information to the nature of the CTC, as    well as the patient's disease. It is possible that staining or image    artifacts may be observed during analysis. For example, enucleated    cells sometimes appear to have a “ghost” region where the nucleus    should have stained, but the corresponding region is nucleic acid    negative. This may be caused by a number of external factors,    including the labeling or imaging techniques. Also, cells have been    observed with “detached” nuclei. While this may possibly indicate a    cell releasing its nucleus, it is more likely that this appears due    to an artifact of the imaging system. However, such “artifacts,”    when real, give valuable information about what may be happening to    the intact cells. Therefore, as part of this invention, suspicious    cells will be more closely analyzed.

Cell fragments are different than “debris” in that debris does notnecessarily resemble a cell. The term debris as used herein, refers tounclassified objects that are specifically or non-specifically labeledduring processing, and are visible as images during analysis, but aredistinct from intact suspect cells. For example, it has been observedthat damaged cells will release nuclear material. During processing,this nuclear material may be non-specifically magnetically labeled, andsubsequently labeled with the nucleic acid stain. During analysis, themagnetically labeled and stained nuclear material can be observed. Thereare other objects that are similarly magnetically selected and stainedwhich appear during analysis that are classified as debris.

The term “morphological analysis” as used herein, refers to visuallyobservable characteristics for an object, such as size, shape, or thepresence/absence of certain features. In order to visualizemorphological features, an object is typically non-specifically stained.The term “epitopical analysis” as used herein, refers to observationsmade on objects that have been labeled for certain epitopes. In order tovisualize epitopic features, an object is typically specifically stainedor labeled. Morphological analysis may be combined with epitopicalanalysis to provide a more complete analysis of an object.

The importance of further visual observation is apparent when fragmentsand debris are often classified as “Not Assigned Events,” or “Unassignedevents”. These terms arise from non-visual analysis, such as with flowcytometry. Because flow cytometry does not image objects, any event notfalling in the specified populations that meet the criteria for thetarget cells, or the non-target cells (as is the case whennon-specifically carried over WBC are negatively labeled), will falloutside either of these populations. However, as will be apparentthroughout this specification, these unassigned events are important.

FIG. 1 is a model of various CTC stages, including shedding andmetastasis. FIG. 1 a shows these stages for cells, clusters, fragments,and debris. FIG. 1 b shows actual images from samples at these samestages. The images of cells clusters, fragments, and debris were takenfrom CellSpotter® analyses of patient samples. The images of tissuesamples (Origin and Metastatic sites) were taken from elsewhere (Manualof Cytology, American Society of Clinical Pathologists Press. 1983).

Briefly, a single cell sheds from a primary tumor into the blood. Thiscell either survives or dies in blood. If it survives, it may possiblydivide in blood, or colonize at a secondary site. If the cell dies,depending on the method, the cell degrades into various types offragments or debris. Another possibility is a cluster of cells is shedfrom a primary tumor into the blood, where it may dissociate into singlecells, or remain intact, and colonize at a secondary site. If thecluster dissociates, it can behave similar to the single cell describedabove. If the cluster remains intact, it is more likely to form asecondary colony for the reasons described above, which includes thelarge diameter cluster becoming lodged in a small diameter capillary.Once lodged, if the cells are viable, the cluster would form a newtumor.

The presence of fragments and debris with very few intact cells suggeststhat there will be little chance of metastasis. Fragmented cells willnot divide, and cannot form secondary tumors. Indeed, only intact CTC orpossibly CTC clusters would be capable of colonizing secondary sites.Identification of antigens that play a role in the adhesion andpenetration process may help. Follow up and assessment of metastaticsites of the patients with and without clusters will also providefurther insight. Nuclear morphology is used to determine the activitystatus and abnormality of a cell. Chromatin clumping, the presence orabsence of nucleoli, and hyperchromasia, are criteria used to determinewhether a cell is benign or malignant, reacting to an immune response,or reacting to treatment. The cytoplasmic morphology is used todetermine the level of differentiation (i.e. tissue of origin). Forexample, cytoplasmic morphology can classify cells as squamous versusglandular.

During blood draw and subsequent specimen processing, the survivingbattered tumor cells present in the peripheral circulation may befurther stressed and damaged by turbulence during blood draw into anevacuated tube and by specimen processing, e.g. transport of the bloodtube and mixing prior to analysis. Such mechanical damage is additionalto on-going immunological, apoptotic, and necrotic in processes leadingto destruction of CTC that occur in vitro in a time dependent manner. Wehave found that the longer the specimen is stored, the greater the lossof CTC, and the larger the amounts of interfering debris and/oraggregates. Indeed, data presented in this specification (FIGS. 2 and 3)show dramatic declines in CTC counts in several blood specimens storedat room temperature or for 24 hrs or longer, indicating substantial invitro destruction of CTC after blood draw. While the losses ofhematopoietic cells are well known phenomena and the subject ofabove-cited patents by Streck Labs and by others, the occurrence ofmechanical damage due to mixing or transport have to date not beenrecognized factors in the loss of CTC or rare cells. The formation ofcellular debris and the interfering effects of accumulating debrisand/or aggregates in the analysis of CTC or other rare cells havesimilarly been unrecognized to date. It appears to be most evident andproblematic in highly sensitive enrichment assays requiring processingof relatively large blood volumes (5-50 mL), and subsequent microscopicdetection or imaging of target cells after volume reduction (less than 1mL). Such debris are either not normally seen, or do not interfere inconventional non-enrichment assays, for example, by flow cytometry or inenrichment by density gradients methods.

To explore if these damaged epithelial cells and epithelial cellfragments observed in patients could be caused by apoptosis of tumorcells induced by chemotherapy, a model to mimic tumor cell death wasdeveloped. Cells of the prostate cell line LnCaP were cultured with orwithout paclitaxel and spiked into blood of healthy donors. Theimmunomagnetically selected cells of the paclitaxel treated samplesanalyzed by CellSpotter® resembled those observed in the patient bloodsamples. Cells treated with paclitaxel displayed signs of apoptosis. Thepunctate cytokeratin staining pattern of the cells appear to correspondwith a collapse of the cytoskeletal proteins (FIG. 4B vs. 6B). Theinitiating event in the sequence resulting from the microtubulestabilizing effects of paclitaxel which in turn may activate thepro-apoptotic gene Bim that senses cytoskeletal distress. Furtherevidence of caspase-cleaved cytokeratin resulting from apoptosis wasobtained with the M30 Cytodeath antibody (Roche Applied Science,Manheim, Germany) that recognizes an epitope of cytokeratin 18 that isonly exposed following caspase cleavage in early apoptosis. Only thepaclitaxel treated LnCaP cells stained with M30 and most of the dimmercytokeratin cells stained with M30, which would be consistent with cellsundergoing apoptosis.

It should be noted that a number of different cell analysis platformscan be used to identify and enumerate cells in the enriched samples.Examples of such analytical platforms are immunicon's CellSpotter®system, a magnetic cell immobilization and analysis system, usingmicroscopic detection for manual observation of cells described inExample 2, and the CellTracks™ system, a more advanced automatic opticalscanning system. These two analytical platforms are described in U.S.Pat. No. 5,876,593; No. 5,985,153 and No. 6,136,182, each of which areincorporated by reference herein as disclosing the respective apparatusand methods for manual or automated quantitative and qualitative cellanalysis.

Other analysis platforms include laser scanning cytometry (Compucyte),bright field base image analysis (Chromavision), and capillary volumetry(Biometric Imaging).

The enumeration of circulating epithelial cells in blood using themethods and compositions of a preferred embodiment of the presentinvention is achieved by immunomagnetic selection (enrichment) ofepithelial cells from blood followed by the analysis of the samples. Theimmunomagnetic sample preparation is important for reducing samplevolume and obtaining as much as a 10⁴ fold enrichment of the target(epithelial) cells. The reagents used for the multi-parameter flowcytometric analysis are optimized such that epithelial cells are locatedin a unique position in the multidimensional space created by thelistmode acquisition of two light scatter and three fluorescenceparameters. These include

-   -   1. an antibody against the pan-leukocyte antigen, CD45 to        identify leukocytes (non-tumor cells);    -   2. a cell type specific or nucleic acid dye which allows        exclusion of residual red blood cells, platelets and other        non-nucleated events; and    -   3. a biospecific reagent or antibody directed against        cytokeratin or an antibody having specificity for an EpCAM        epitope which differs from that used to immunomagnetically        select the cells.

It will be recognized by those skilled in the art that the method ofanalysis of the enriched tumor cell population will depend on theintended use of the invention. For example, in screening for cancers ormonitoring for recurrence of disease, as described hereinbelow, thenumbers of circulating epithelial cells can be very low. Since there issome “normal” level of epithelial cells, (very likely introduced duringvenipuncture), a method of analysis that identifies epithelial cells asnormal or tumor cells is desirable. In that case, microscopy basedanalyses may prove to be the most accurate. Such examination might alsoinclude examination of morphology, identification of known tumordiathesis associated molecules (e.g., oncogenes).

Patients

Patients' age range was 47-91 year (mean 74), with initial diagnosis 2to 10 years prior to study. Medical records were reviewed for therapyand stage. Patients and healthy volunteers signed an informed consentunder an approved research study. Blood was drawn into 10 ml EDTAVacutainer tubes (Becton-Dickinson, NJ). Samples were kept at roomtemperature and processed within 6 hours after collection unlessindicated otherwise.

Sample Preparation

Magnetic nanoparticles labeled with monoclonal antibodies identifyingepithelial cell adhesion molecule (EpCAM) were used to label andseparate by magnetic means epithelial cells from hematopoietic cells, astaught in commonly-owned U.S. Pat. No. 6,365,362, and U.S. patentapplication Ser. No. 10/079,939, filed 19 Feb. 2002, both of which arefully incorporated by reference herein. The magnetically captured cellsresuspended in a volume of 200 μl are fluorescently labeled todifferentiate between hematopoietic and epithelial cells. A monoclonalantibody that recognizes keratins 4, 5, 6, 8, 10, 13, and 18, conjugatedto Phycoerythrin (CK-PE) was used to identify epithelial cells and amonoclonal antibody that recognizes CD45 was used to identify leukocytesand identify hematopoietic cells that non-specifically bind tocytokeratin.

For multicolor fluorescent microscopy (CellSpotter®) analysis CD45 wasconjugated to allophycocyanin (CD45-APC, Caltag, CA) whereas for flowcytometric analysis perdinin chlorophyll protein conjugated CD45(CD45-PerCP, BDIS San Jose, Calif.) was used. The nucleic acid specificdye DAPI (4,6-diamidino-2-phenylindole) was used to identify andvisualize the nucleus with the CellSpotter® system and the nucleic aciddye used in the Procount system (BDIS, San Jose, Calif.) was used toidentify cells by flow cytometry. After incubation, the excess stainingreagents were aspirated and discarded and the captured cells wereresuspended and transferred into a 12×75 mm tube for flow cytometricanalysis or to a CellSpotter® analysis chamber (as described in U.S.application Ser. No. 10/074,900, filed 12 Feb. 2002, incorporated byreference herein) contained within a magnetic yoke assembly that holdsthe chamber between two magnets (Captivate, Molecular Probes, OR).

EXAMPLE 1 Sample Analysis via Flow Cytometry

Samples were analyzed on a FACSCalibur flow cytometer equipped with a488 nm argon ion laser (BDIS, San Jose, Calif.). Data acquisition wasperformed with CellQuest (BDIS, San Jose, Calif.) using a threshold onthe fluorescence of the nucleic acid dye. The acquisition was haltedafter 8000 beads or 80% of the sample was analyzed. Multiparameter dataanalysis was performed on the listmode data (Paint-A-Gate^(pro), BDIS,San Jose, Calif.). Analysis criteria included size defined by forwardlight scatter, granularity defined by orthogonal light scatter, positivestaining with the PE-labeled anti-cytokeratin MAb and no staining withthe PerCP-labeled anti-CD45 Mab. For each sample, the number of eventspresent in the region typical for epithelial cells was multiplied by1.25 to account for the sample volume not analyzed by flow cytometry.

FIG. 2 Panels A, B and C shows the flow cytometric analysis of a bloodsample of a patient with metastatic prostate cancer. Two vertical linesin Panel B illustrate the low and high boundary of nucleic acid (NAD)content of leukocytes (red dots). CTC candidates express Cytokeratin(CK+), lack CD45 (CD45−) and contain nucleic acids (NAD+). CTCcandidates having NAD equal or higher than leukocytes are consideredcells and are depicted black. CK+, CD45− events with NAD content lessthan leukocytes were not considered cells and depicted blue. The blueevents were clearly smaller as compared with the black colored CTC asevident by the smaller forward light scatter signals. The threshold onthe NAD staining intensity clearly excluded a large portion of CK+,CD45− events with even lower NAD staining intensity. In analysis ofblood samples from healthy donors few such CK+, CD45− events areobserved suggesting that this phenomenon is related to cancer. A typicalexample of an analysis of a blood sample from a healthy donor is shownin FIGS. 2D, 2E, and 2F.

EXAMPLE 2 Sample Analysis via CellSpotter®

The CellSpotter® system consists of a microscope with a Mercury Arc Lampmercury arc lamp, a 10× objective, a high resolution X, Y, Z stage and afour filter cube changer. Excitation, dichroic and emission filters ineach of four cubes were for DAPI 365 nm/400 nm/400 nm, for DiOC16 480nm/495 nm/510 nm, for PE 546 nm/560 nm/580 nm and for APC 620 nm/660nm/700 nm. Images were acquired with a digital camera connected to adigital frame grabber. The surface of the chamber is 80.2 mm² and 4 rowsof 35 images for each of the 4 filters resulting in 560 images have tobe acquired to cover the complete surface. The CellSpotter® acquisitionprogram automatically determines the region over which the images are tobe acquired, the number of images to acquire, the position of each imageand the microscope focus to use at each position. All the images from asample are logged into a directory that is unique to the specific sampleidentification. An algorithm is applied on all of the images acquiredfrom a sample to search for locations that stain for DAPI and CK-PE. Ifthe staining area is consistent with that of a potential tumor cell(DAPI+, CK-PE+) the software stores the location of these areas in adatabase. The software displays thumbnails of each of the boxes and theuser can confirm that the images represented in the row are consistentwith tumor cells, or stain with the leukocyte marker CD45. The softwaretabulates the checked boxes for each sample and the information isstored in the database.

FIG. 3 shows examples of CellSpotter® analysis of a blood sample from apatient with metastatic prostate cancer. Regions that potentiallycontain tumor cells are displayed in rows of thumbnails. The ruler inthe left lower corner of the figure indicates the sizes of thethumbnails. From right to left these thumbnails represent nuclear(DAPI), cytoplasmic cytokeratin (CK-PE), control cell (DiOC₁₆(3)) andsurface CD45 (CD45-APC) staining. The composite images shown at the leftshow a false color overlay of the purple nuclear (DAPI) and greencytoplasmic (CK-PE) staining. The check box beside the composite imageallow the user to confirm that the images represented in the row areconsistent with tumor cells and the check box beside the CD45-APC imageis to confirm that a leukocyte or tumor cell stain non-specifically. Inthis patient sample, the software detected 2761 rows of thumbnails thatdemonstrated staining consistent with tumor cells. Eighteen of the 2761rows are shown in the figure labeled 1631-1640 and 1869-1876. Rowsnumbered 1631, 1636, 1638, 1640, and 1873-1876 are checked off anddisplay features of CTC defined as a size greater than 4 μm, thepresence of a nucleus surrounded by, cytoplasmic cytokeratin stainingand absence of DiOC₁₆(3) and CD45 staining. Note the difference inappearance of the tumor cells: the cell in row 1638 is large and the onein row 1640 is significantly smaller. The immunophenotype of the eventsin rows 1634 and 1869 are consistent with tumor cells but theirmorphology is not consistent with intact cells. The thumbnail in row1869 shows a large nucleus and speckled cytoplasmic due to retraction ofcytoskeletal proteins consistent with apoptosis of the cell. Thethumbnail in row 1634 shows a damaged cell that appears to, extrude itsnucleus. The thumbnail shown in row 1632 shows a cell that stains bothwith cytokeratin as well as CD45 and is either a tumor cellnon-specifically binding to CD45 or a leukocyte non specificallystaining with cytokeratin. In this instance the morphology of the cellclosely resembles that of a lymphocyte. The thumbnails shown in rows1633, 1635, 1637, 1639, 1870 and 1872 shows cytokeratin staining objectsthat are larger that 4 μm but have no resemblance to cells. Thecytokeratin staining objects in thumbnails 1637, 1639 and 1872 are inclose proximity of a leukocyte.

Based on observation of images of CTC candidates in several patientsamples, CTC were classified into three categories: intact CTC, damagedCTC, and CTC fragments all not staining with CD45 and not appearing inthe DiOC₁₆(3) filter. FIG. 4 displays examples of the three categoriesof CTC isolated from a single tube of blood of a patient with metastaticprostate cancer undergoing therapy. Intact tumor cells shown in FIG. 3Awere defined as objects larger than 4 μm with a relatively smoothcytoplasmic membrane, cytoskeletal proteins throughout the cytoplasm,and an intact nucleus encompassed within the nucleus. Damaged CTC shownin FIG. 4B were defined as objects larger than 4 μm with speckledcytokeratin staining or ragged cytoplasmic membrane, and a nucleusassociated with the cytokeratin staining. Tumor cell fragments shown inFIG. 4C were defined as round cytokeratin staining objects larger than 4μm with or without association of nuclear material that had nomorphological resemblance to a cell.

EXAMPLE 3 CTC in Prostate Cancer Patients

CTC were enumerated in 18 blood samples of prostate cancer patients and27 samples from healthy individuals by both flow cytometry andCellSpotter®. The results shown in Table 1 were sorted by increasingnumber of intact CTC detected by CellSpotter®.

TABLE 1 Enumeration of CTC by CellSpotter ® and flow cytometry in 18blood samples of prostate cancer patients and 27 samples from healthyindividuals. Flow CellSpotter ® Cytometry Intact Tumor Damaged TumorTumor Cell CK + CD45 − Patient Cells Cells Fragments NA + Sample # % # %# % # 1 0 0 1 50 1 50 5 2 0 0 2 100 0 0 12 3 0 0 2 66 1 34 1 4 0 0 2 502 50 0 5 0 0 2 29 5 71 5 6 0 0 3 60 2 40 18 7 0 0 3 38 5 62 0 8 0 0 7 449 56 10 9 0 0 13 76 4 24 2 10 1 5 1 5 20 90 4 11 1 10 4 40 5 50 0 12 222 1 11 6 67 4 13 28 6 7 1 441 93 69 14 70 5 168 12 1204 83 683 15 322 3448 13 4244 87 500 16 350 5 112 2 5924 93 723 17 350 2 1429 9 14412 892420 18 742 17 112 2 3641 81 310 Mean — 4% — 34% — 62% — 27 samples fromhealthy donors Mean 0.04 0.96 4.96 0.7 SD 0.19 1.85 3.98 1.14 Min 0 0 00 Max 1 7 15 4 # - number CTC in 7.5 ml blood % - percentage of all CTCdetected by CellSpotter ®

In the CellSpotter® analysis, the proportion of intact CTC clearlyconstituted the smallest fraction of CTC and ranged from 0% to 22% ofall CTC (mean 4%). The proportion of damaged CTC ranged from 1% to 100%(mean 34%) and the CTC fragments constituted the largest portion of CTCranging from 0% to 93% (mean 62%). The distribution of CTC over thethree categories between the patients varied considerably as amplifiedby a lack of correlation between intact CTC and damaged CTC (R²=0.20)and intact CTC and CTC fragments (R²=0.42) and some correlation betweendamaged CTC and CTC fragments (R²=0.88). Comparison of intact CTC byCellSpotter® and CTC enumerated by flow cytometry showed no significantcorrelation (R²=0.26) whereas significant correlations were foundbetween the damaged CTC and CTC by flow cytometry (R²=0.92) and CTCfragments and CTC by flow cytometry (R²=0.93). Comparison of the CTCdetected by flow cytometry and CellSpotter® suggests that CTC detectedby flow cytometry encompass intact CTC as well as damaged CTC and to acertain extent, CTC fragments.

EXAMPLE 4 Mimicking Cell Damage by In-Vitro Induction of Apoptosis inLncaP Cells

To investigate the effect of apoptosis induced by cytotoxic agents onflow cytometric and CellSpotter® analysis of CTC, cells from theprostate cell line LnCaP were cultured in the presence or absence of 40nM paclitaxel for 72 hours. Following incubation, untreated LnCaP cellsdemonstrated a viability of >95% by trypan blue exclusion and 33% forthe paclitaxel treated cells. The treated and untreated LnCaP cells werespiked into blood of healthy donors, selected by the ferrofluid methodsdescribed above, and analyzed by the CellSpotter® system. In experimentsin which LnCaP cells were spiked into blood that were not treated withpaclitaxel greater than 95% of the LnCaP cells were classified as intacttumor cells. The morphologic appearance of the paclitaxel treated LnCaPcells showed close resemblance to that of the CTC observed in thepatient samples and are shown in FIG. 6. Intact LnCaP cells thatsurvived paclitaxel treatment are shown in FIG. 6A, damaged LnCaP, ofwhich the majority show speckled cytokeratin staining, are shown in FIG.6B, and tumor fragments are shown in FIG. 6C.

Normal blood samples spiked with paclitaxel treated and untreated LnCaPcells were also prepared for flow cytometric analysis. In FIGS. 2G, 2H,and 21, the flow cytometric analysis of a blood sample spiked with 501LnCaP cells is shown. A predominantly bright cytokeratin positivepopulation with a nucleic acid content greater than normal humanleukocytes and relatively large size as illustrated by the large forwardlight scatter signals is shown and depicted black in the figure. Onlyfew CK+, CD45− events with NAD content less than leukocytes and depictedblue are detected in the sample. FIGS. 2J, 2K, and 2L shows the flowcytometric analysis of paclitaxel treated LnCaP cells spiked in blood.In contrast to viable LnCaP cells, a wide distribution of cytokeratinstaining was observed with a significant portion of the populationdemonstrating a decreased concentration of nucleic acid content. Inaddition, numerous small cytokeratin positive events with less nucleicacid content as leukocytes were observed. The pattern of the patientclosely resembled that of the pattern of the paclitaxel treated LnCaPcells supporting the hypothesis that the CTC detected by flow cytometryrepresent intact CTC as well as a variety of disintegrating cells inblood of cancer patients.

The data shown above demonstrate that in the blood of patients withprostate cancer, CTC detected by both flow cytometry and CellSpotter®are comprised of intact cells and a variety of disintegrated cells. Theapoptosis induced in vitro by paclitaxel suggests that the detected CTCin patient blood samples are undergoing apoptosis, necrosis, or in vivodamage to a varying degree caused by the treatment, mechanical damage bypassage through the vascular system, or by the immune system.

Another source of cell disintegration caused in vitro could, however, beintroduced by the sample preparation or the lack of stability of CTC orother blood components after blood draw. To investigate the effect ofsample aging, blood samples drawn from 12 patients with prostate cancerwere processed and analyzed by flow cytometry within two hours, after 24hours, and after 6 and 18 hours if sufficient blood was available. In 8of the 12 patient samples, CTC were detected at a level greater than themean +3SD of that detected in normal donors. As shown in Table 2, a lossof CTC with sample aging was observed in all 8 samples.

TABLE 2 Enumeration of CTC by flow cytometry in 8 blood samples ofprostate cancer patients processed and analyzed at different time pointsafter blood draw Time after blood draw <2 hr ~6 hr ~18 hr ~24 hr Patient# #CTC #CTC #CTC #CTC 1 5 — — 0 2 8  9  2 3 3 15 — — 0 4 31 — — 3 5 44 —— 8 6 45 — — 1 7 49 38 19 26 8 78 — — 0 hr = hours #CTC = number of CTCin 5 ml blood

Significant reductions in the number of CTC were detected when bloodprocessing was delayed demonstrating the fragility of CTC, and making itnecessary to process non-stabilized blood samples no later than sixhours after blood draw to obtain accurate CTC counts. To reliably assessif clinically relevant information is contained within the differentstages of tumor cell degradation, a blood preservative is needed thatstabilizes CTC at the time of blood draw to obtain an accuratereflection of what is occurring inside the body. Furthermore, the samplepreparation method for sensitive assays used to enrich for CTC requiresthat all classes of CTC are captured, and therefore excludes the use oftraditional density gradient separation methods in the prior art.

EXAMPLE 5 Obvious CTC and Suspect CTC are Important Indicators

It is important to be able to distinguish between in vivo and in vitrodamage for sensitive assays, such as those described here. This isespecially evident when the assay attempts to determine theeffectiveness of treatments or therapies, which are known to cause invivo cellular damage. If sample handling, processing, or analysis wereto result in damaging the target cells, forming suspect cells,fragments, or debris, the assay will not give meaningful results.

An assay was developed to directly detect CTC in 100 μl of blood withoutany enrichment method with a flow cytometer. The 100 μl assay detectsonly EpCAM positive cells and the sensitivity is very low. However, someadvanced stage cancer patients with high CTC counts are expected to beobservable. This assay should give a reliable confirmatory estimation ofCTC because it is a direct assay that involves no manipulation. Data wasgenerated with several patient samples using the assay to answer severalquestions.

The 100 μl assay categorizes cells based on properties such as size andstaining intensity. Obvious CTC have bright nucleic acid staining(similar to leukocytes), positive EpCAM antigen staining and sizesimilar to leukocytes or larger. Suspect CTC are any objects positivefor EpCAM antibody but not characterized as obvious CTC (i.e. dimnucleic acid, size smaller than leukocytes). The assay identifiesobjects from both categories.

FIG. 5 shows the presence of obvious and suspect CTC in blood asdetermined by the 100 μl assay. The suspect CTC are not created duringsample processing (in vitro damage) as the 100 μl assay is a directassay and does not involve any separation or wash steps. The data abovealso show there is a relationship between the number of obvious andsuspect CTC. The number of suspect CTC seems to increase as the numberof obvious CTC increases. When the numbers of suspect versus obvious CTCis plotted, the slope of 2.92 indicates the proportion of suspect CTCpresent in sample when compared to obvious CTC. The correlationcoefficient of r²=0.97 shows an excellent correlation between obviousCTC and suspect CTC for a number of clinical samples. In addition,suspect CTC are also seen in the ferrofluid-selection assay, and haveproperties similar to suspect CTC detected in the blood by the directassay. It is important to include suspect CTC in addition to obvious CTCin total tumor cell count.

An important question is how the data from the 100 μl assay compareswith ferrofluid-selected CTC (enriched CTC). Does the assayquantitatively detect CTC? Another question is what is the recovery ofCTC in the ferrofluid-selected assay if the flow assay data is correct.Three main factors determining the recovery of CTC in the 100 μl assayare:

-   -   EpCAM density,    -   cytokeratin positivity, and    -   nucleus positivity.        The suspect CTC have lower EpCAM density compared to obvious CTC        and significance of this is not yet well known.

A comparison was made of obvious and suspect CTC by the 100 μl assay tothe ferrofluid-selection assay using 7 ml of blood. This data wasobtained from a prostate patient samples and analyzed by flow cytometry.Both obvious and suspect CTC increased with storage time and the trendwas similar to CTC detected in the ferrofluid-selection assay, therebyvalidating the 100 μl assay. The recovery of CTC from theferrofluid-selection assay was about 90% based on the CTC in 100 μl ofblood. It was also known that MFI (Mean Fluorescence Intensity whichcorrelates the EpCAM density) of CTC from this patient was high(MFI=300), and all EpCAM positive cells are cytokeratin positive.However, the recoveries of CTC from some other clinical samples has beenas low as 20%. There may be several factors that contribute for a lowerrecovery, such as EpCAM positive/cytokeratin negative cells, cytokeratindim cells, and mucin on the cell surface inhibiting the ability offerrofluid to bind cells.

The assay described herein was performed on patients at two times.Response was measured by bi-dimensional imaging of the lesion. TheRation (Ratio=Obvious CTC/Total CTC) is similar to the Response Index,described earlier, and can be used as a numeric indicator of treatmentsuccess. The results are summarized in Table 3. Ratios near 1.0 indicatethe Total CTC are obvious CTC, and ratios near 0.0 indicate more suspectCTC or debris. Progression indicates the lesion is increasing in size,Partial Response indicates a response to treatment where the Ratio isrelatively low, and stabilized indicates no change, or reduction inlesion size. A positive change indicates an increase in the number ofintact CTC, corresponding to the progression of the disease. A negativechange indicates a decrease in the number of intact CTC, or a possibleincrease in the number of suspect CTC and/or debris, corresponding to aresponse to treatment.

These results show the importance of including suspect CTC and debriswhen analyzing response to treatment because the numbers of intact orobvious CTC alone would not provide as much information. Furthermore,such indicators are useful for short-term monitoring of treatments andtherapies, or longer term monitoring for remission and/or relaps.

TABLE 3 Obvious CTC and suspect CTC corresponding to a treatmentresponse Response Ratio1 Ratio2 Change Progressive 0.3 0.0 −0.3 0.0 0.00.0 0.5 0.6 0.1 0.9 1.0 0.1 0.3 0.5 0.2 0.4 0.7 0.3 0.0 0.4 0.4 0.5 0.90.4 0.0 0.5 0.5 0.0 0.6 0.6 Partial 1.0 0.0 −1.0 Response 0.4 0.0 −0.40.3 0.0 −0.3 0.5 0.2 −0.3 0.4 0.3 −0.1 0.0 0.0 0.0 0.3 1.0 0.7Stabilized 1.0 0.0 −1.0 0.5 0.0 −0.5 1.0 0.7 −0.3 0.3 0.0 −0.3 0.4 0.3−0.1 0.1 0.0 −0.1 0.2 0.1 −0.1 0.6 0.5 −0.1 0.9 0.8 −0.1 0.0 0.0 0.0 0.60.7 0.1 0.6 0.8 0.2 0.0 1.0 1.0

Enumeration of tumor cell debris may prove more significant in cancerdiagnostics and therapeutics than detection of large proliferative cellclusters. Since debris particles in the size range, probably about 1-3μm (the size of platelets), have been observed to be present in muchlarger amounts than intact cells, they may constitute a separate,independent, and possibly more sensitive marker than intact tumor cells.The presence of damaged CTC may be particularly relevant in detectingearly-stage cancer when the immune system is intact and most active.Similarly, dramatic increases in debris during therapy may suggestbreakdown of both circulating and tissue tumor cells (i.e. therapeuticeffectiveness), paralleling the massive release of cellular componentslike calcium observed during tumor disintegration. Like soluble tumormarkers, such debris may be detectable in blood without enrichment, orwith minimal enrichment in the buffy coat layer and constitute analternative, and potentially simpler diagnostic tool than intact cellenrichment/analysis. Since morphology is lost in CTC debris, detectioncould be done by flow cytometry as long as the debris is stained for theappropriate determinants, such as cytokeratin.

As previously discussed, damaged or fragmented CTC with or without DNAare theoretically to be expected, and therefore are not undesirableevents in specimens from patients undergoing effective therapy and inuntreated patients with strong immune systems. The ratio or % of intactCTC to total detectable events may prove to be a useful parameter to theclinician in assessing a patient's immune system or response to therapy.The normal immune defenses, especially activated neutrophils, also candamage or destroy CTC as foreign species by a process called“extracellular killing” even if the CTC are larger than the neutrophils.It does not seem surprising to find only a small percentage of the shedCTC as intact cells, unless the immune system is overwhelmed in the latestages of disease or therapy is ineffective.

Hence, there are a number of methods for in vitro cancer detection:conclusive detection of intact circulating cells/clusters, andinferential methods like circulating tumor debris (including total andtumor-specific RNA/DNA, and conventional soluble tumor markers).However, no method by itself may be sufficiently sensitive. Lowerspecificity of debris detection compared to CTC morphology may be aproblem in screening that could be minimized (e.g. with triplelabeling), but it may be a lesser problem in monitoring. Furtherstatistical analysis and correlations on debris data relative to intactCTC and diagnostic stage in patients compared to normals appearworthwhile in assessing the sensitivity and specificity of debrisanalysis.

Examples of different types of cancer that may be detected using thecompositions, methods and kits of the present invention include apudoma,choristoma, branchioma, malignant carcinoid syndrome, carcinoid heartdisease, carcinoma e.g., Walker, basal cell, basosquamous, Brown-Pearce,ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous,non-small cell lung, oat cell, papillary, scirrhous, bronchiolar,bronchogenic, squamous cell and transitional cell reticuloendotheliosis,melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma,fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma,mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing'ssarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma,chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma,cementoma, odontoma, teratoma, throphoblastic tumor, adenocarcinoma,adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma,cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma,hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertolicell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma,myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma,ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma,neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma,paraganglioma nonchromaffin, antiokeratoma, angioma sclerosing,angiomatosis, glomangioma, hemangioendothelioma, hemangioma,hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma,cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma,leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma,ovarian carcinoma, rhabdomyosarcoma, sarcoma (Kaposi's, and mast-cell),neoplasms (e.g., bone, digestive system, colorectal, liver, pancreatic,pituitary, testicular, orbital, head and neck, central nervous system,acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis,and cervical dysplasia.

However, the present invention is not limited to the detection ofcirculating epithelial cells and/or clusters, fragments, or debris only.For example, endothelial cells have been observed in the blood ofpatients having a myocardial infarction. Endothelial cells, myocardialcells, and virally infected cells, like epithelial cells, have cell typespecific determinants recognized by available monoclonal antibodies.Accordingly, the methods and the kits of the invention may be adapted todetect such circulating endothelial cells. Additionally, the inventionallows for the detection of bacterial cell load in the peripheral bloodof patients with infectious disease, who may also be assessed using thecompositions, methods and kits of the invention. It would be reasonableto expect that these rare cells will behave similarly in circulation,and that fragments and/or debris will be present in similar conditionsas those described hereinabove.

The preferred embodiments of the invention as herein disclosed, are alsobelieved to enable the invention to be employed in fields andapplications additional to cancer diagnosis. It will be apparent tothose skilled in the art that the improved diagnostic modes of theinvention are not to be limited by the foregoing descriptions ofpreferred embodiments. Finally, while certain embodiments presentedabove provide detailed descriptions, the following claims are notlimited in scope by the detailed descriptions. Indeed, variousmodifications may be made thereto without departing from the spirit ofthe following claims.

1. A method for diagnosing disease in a test subject comprising: a.obtaining a biological specimen from a test subject, said specimencomprising a mixed cell population suspected of containing intact rarecells and further comprising: i. cell fragments derived from rare cells,or ii. cellular debris derived from rare cells; b. preparing amagnetically-labeled sample wherein said biological sample is mixed withmagnetic particles coupled to a first biospecific ligand which reactsspecifically with said intact rare cells, and said cell fragments orsaid cellular debris, to the substantial exclusion of other specimencomponents; c. contacting said magnetically-labeled sample with at leastone additional biospecific ligand which specifically labels said intactrare cells, and said cell fragments or said cellular debris, to thesubstantial exclusion of other specimen components; d. analyzing saidlabeled rare cells, and said labeled cell fragments or said labeledcellular debris, the presence of said labeled rare cells, said labeledcell fragments, and said labeled cellular debris indicating the presenceof disease.
 2. The method of claim 1, wherein said biological specimenis blood.
 3. The method of claim 2, wherein after said biologicalspecimen obtained, it is contacted with an agent capable of stabilizingsaid biological specimen.
 4. The method of claim 1, wherein saidmagnetic particles are colloidal.
 5. The method of claim 1, whereinafter the step of preparing said magnetically-labeled sample, saidsample is subjected to a high gradient magnetic field to produce aseparated magnetically-labeled fraction which is enriched for saidintact rare cells, and said cell fragments or said cellular debris. 6.The method of claim 1, wherein said analysis is selected from the groupconsisting of: multiparameter flow cytometry, immunofluorescentmicroscopy, laser scanning cytometry, bright field base image analysis,capillary volumetry, spectral imaging analysis, manual cell analysis,and automated cell analysis.
 7. The method of claim 1, wherein saidanalysis is based on at least one of the group consisting of:morphologic analysis and epitopic analysis.
 8. A method for diagnosingdisease in a test subject comprising: a. obtaining a biological specimenfrom a test subject, said specimen comprising a mixed cell populationsuspected of containing intact rare cells and clusters of rare cells; b.preparing a magnetically-labeled sample wherein said biological sampleis mixed with magnetic particles coupled to a first biospecific ligandwhich reacts specifically with said intact rare cells and said clustersof rare cells, to the substantial exclusion of other specimencomponents; c. contacting said magnetically-labeled sample with at leastone additional biospecific ligand which specifically labels said intactrare cells and said clusters of rare cells, to the substantial exclusionof other specimen components; d. analyzing said labeled rare cells andsaid labeled clusters of rare cells, the presence of said labeled rarecells and said labeled clusters of rare cells indicating the presence ofdisease.
 9. The method of claim 8, wherein said biological specimen isblood.
 10. The method of claim 9, wherein after said biological specimenobtained, it is contacted with an agent capable of stabilizing saidbiological specimen.
 11. The method of claim 8, wherein said magneticparticles are colloidal.
 12. The method of claim 8, wherein after thestep of preparing said magnetically-labeled sample, said sample issubjected to a high gradient magnetic field to produce a separatedmagnetically-labeled fraction which is enriched for said intact rarecells and said clusters of rare cells.
 13. The method of claim 8,wherein said analysis is selected from the group consisting of:multiparameter flow cytometry, immunofluorescent microscopy, laserscanning cytometry, bright field base image analysis, capillaryvolumetry, spectral imaging analysis, manual cell analysis, andautomated cell analysis.
 14. A method for diagnosing malignancy in atest subject comprising: a. obtaining a biological specimen from a testsubject, said specimen comprising a mixed cell population suspected ofcontaining intact malignant cells and further comprising: i. cellfragments derived from malignant cells, or ii. cellular debris derivedfrom malignant cells; b. preparing a magnetically-labeled sample whereinsaid biological sample is mixed with magnetic particles coupled to afirst biospecific ligand which reacts specifically with said intactmalignant cells, and said cell fragments or said cellular debris, to thesubstantial exclusion of other specimen components; c. contacting saidmagnetically-labeled sample with at least one additional biospecificligand which specifically labels said intact malignant cells, and saidcell fragments or said cellular debris, to the substantial exclusion ofother specimen components; d. analyzing said labeled malignant cells,and said labeled cell fragments or said labeled cellular debris, thepresence of said labeled malignant cells, said labeled cell fragments,and said labeled cellular debris indicating the presence of malignancy.15. The method of claim 14, wherein said biological specimen is blood.16. The method of claim 15, wherein after said biological specimenobtained, it is contacted with an agent capable of stabilizing saidbiological specimen.
 17. The method of claim 14, wherein said magneticparticles are colloidal.
 18. The method of claim 14, wherein after thestep of preparing said magnetically-labeled sample, said sample issubjected to a high gradient magnetic field to produce a separatedmagnetically-labeled fraction which is enriched for said intactmalignant cells, and said cell fragments or said cellular debris. 19.The method of claim 14, wherein said analysis is selected from the groupconsisting of: multiparameter flow cytometry, immunofluorescentmicroscopy, laser scanning cytometry, bright field base image analysis,capillary volumetry, spectral imaging analysis, manual cell analysis,and automated cell analysis.
 20. The method of claim 14, wherein saidanalysis further comprises classifying cell fragments or said cellulardebris based on their origin as caused by apoptosis or necrosis.
 21. Themethod of claim 20, wherein analysis further comprises classifying cellfragments or said cellular debris based on their origin as caused bymechanical damage, drug-induced damage, or immunological damage.
 22. Themethod of claim 20, wherein said classification is based on at least oneof the group consisting of: morphologic analysis and epitopic analysis.23. A method for diagnosing malignancy in a test subject comprising: a.obtaining a biological specimen from a test subject, said specimencomprising a mixed cell population suspected of containing intactmalignant cells and clusters of malignant cells; b. preparing amagnetically-labeled sample wherein said biological sample is mixed withmagnetic particles coupled to a first biospecific ligand which reactsspecifically with said intact malignant cells and said clusters ofmalignant cells, to the substantial exclusion of other specimencomponents; c. contacting said magnetically-labeled sample with at leastone additional biospecific ligand which specifically labels said intactmalignant cells and said clusters of malignant cells, to the substantialexclusion of other specimen components; d. analyzing said labeledmalignant cells and said labeled clusters of malignant cells, thepresence of said labeled malignant cells and said labeled clusters ofmalignant cells indicating the presence of malignancy.
 24. The method ofclaim 23, wherein said biological specimen is blood.
 25. The method ofclaim 24, wherein after said biological specimen obtained, it iscontacted with an agent capable of stabilizing said biological specimen.26. The method of claim 23, wherein said magnetic particles arecolloidal.
 27. The method of claim 23, wherein after the step ofpreparing said magnetically-labeled sample, said sample is subjected toa high gradient magnetic field to produce a separatedmagnetically-labeled fraction which is enriched for said intactmalignant cells and said clusters of malignant cells.
 28. The method ofclaim 23, wherein said analysis is selected from the group consistingof: multiparameter flow cytometry, immunofluorescent microscopy, laserscanning cytometry, bright field base image analysis, capillaryvolumetry, spectral imaging analysis, manual cell analysis, andautomated cell analysis.
 29. A kit for assaying a biological specimenfor the presence of malignant cells, and cell fragments derived frommalignant cells or cellular debris derived from malignant cells,comprising: a. coated magnetic nanoparticles comprising: i. a magneticcore material, ii. a protein base coating material, and iii. an antibodythat binds specifically to a first characteristic determinant of saidmalignant cell, and said cell fragments or said cellular debris, whereinsaid antibody is coupled to said base coating material; b. at least oneantibody having binding specificity for a second characteristicdeterminant of said malignant cell, and said cell fragments or saidcellular debris; c. an agent capable of staining further features ofsaid malignant cells, and said cell fragments or said cellular debris.30. The kit of claim 59, further comprising a panel of antibodies eachspecific for a different characteristic determinant.
 31. The kit ofclaim 59, further comprising a specific agent capable of labelingnon-target entities.